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The species Metchnikovella dogieli (Paskerova et al. Protistology 10:148-157, 2016) belongs to one of the early diverging microsporidian groups, the metchnikovellids (Microsporidia: Metchnikovellidae). In relation to typical ('core') microsporidia, this group is considered primitive. The spores of metchnikovellids have no classical polar sac-anchoring disk complex, no coiled polar tube, no posterior vacuole, and no polaroplast. Instead, they possess a short thick manubrium that expands into a manubrial cistern. These organisms are hyperparasites; they infect gregarines that parasitise marine invertebrates. M. dogieli is a parasite of the archigregarine Selenidium pygospionis (Paskerova et al. Protist 169:826-852, 2018), which parasitises the polychaete Pygospio elegans. This species was discovered in samples collected in the silt littoral zone at the coast of the White Sea, North-West Russia, and was described based on light microscopy. No molecular data are available for this species, and the publicly accessible genomic data for metchnikovellids are limited to two species: M. incurvata Caullery & Mesnil, 1914 and Amphiamblys sp. WSBS2006. In the present study, we applied single-cell genomics methods with whole-genome amplification to perform next-generation sequencing of M. dogieli genomic DNA. We performed a phylogenetic analysis based on the SSU rRNA gene and reconstructed a multigene phylogeny using a concatenated alignment that included 46 conserved single-copy protein domains. The analyses recovered a fully supported clade of metchnikovellids as a basal group to the core microsporidia. Two members of the genus Metchnikovella did not form a clade in our tree. This may indicate that this genus is paraphyletic and requires revision.

Representatives of Apicomplexa perform various kinds of movements that are linked to the different stages of their life cycle. Ancestral apicomplexan lineages, including gregarines, represent organisms suitable for research into the evolution and diversification of motility within the group. The vermiform trophozoites and gamonts of the archigregarine Selenidium pygospionis perform a very active type of bending motility. Experimental assays and subsequent light, electron, and confocal microscopic analyses demonstrated the fundamental role of the cytoskeletal proteins actin and tubulin in S. pygospionis motility and allowed us to compare the mechanism of its movement to the gliding machinery (the so-called glideosome concept) described in apicomplexan zoites. Actin-modifying drugs caused a reduction in the movement speed (cytochalasin D) or stopped the motility of archigregarines completely (jasplakinolide). Microtubule-disrupting drugs (oryzalin and colchicine) had an even more noticeable effect on archigregarine motility. The fading and disappearance of microtubules were documented in ultrathin sections, along with the formation of α-tubulin clusters visible after the immunofluorescent labelling of drug-treated archigregarines. The obtained data indicate that subpellicular microtubules most likely constitute the main motor structure involved in S. pygospionis bending motility, while actin has rather a supportive function.

Gregarines are a common group of parasites that infect the intestines of marine invertebrates, and particularly polychaetes. Here, we describe for the first time four gregarine species that inhabit the intestines of three spionid species: Dipolydora cf. flava, Spio quadrisetosa and Boccardia proboscidea from the Patagonian coast, Argentina, using light and scanning electron microscopy and molecular phylogenetic analyses of small subunit (SSU) rDNA sequences. Even though the spionid species thrive in the same environments, our results showed a high host specificity of the gregarine species. Selenidium cf. axiferens and Polyrhabdina aff. polydorae were both identified from the intestine of D. cf. flava. The new species, Polyrhabdina madrynense sp. n. and Selenidium patagonica sp. n., were described from the intestines of S. quadrisetosa and the invasive species B. proboscidea, respectively. All specimens of D. cf. flava and S. quadrisetosa were infected by gregarines (P = 100%), recording the highest mean intensity values of infection (MI = 80; 60 respectively), in contrast to B. proboscidea (P = 60%; MI = 38). We associated this finding with the recent invasion of this host. It is expected that in the future, an increase of its population density might favour a rising intensity of this gregarine infection. Fil: Rueckert, S.. Edinburgh Napier University; Reino Unido Fil: Glasinovich, Natusha Irina. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Centro Nacional Patagónico. Instituto de Biología de Organismos Marinos; Argentina Fil: Diez, María Emilia. Universidad Nacional de la Patagonia "San Juan Bosco"; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina Fil: Cremonte, Florencia. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Centro Nacional Patagónico. Instituto de Biología de Organismos Marinos; Argentina Fil: Vázquez, Nuria Natalia. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Centro Nacional Patagónico. Instituto de Biología de Organismos Marinos; Argentina

Described here is a polymerase chain reaction (PCR) test to detect the apicomplexan-X (APX) parasite of a flat oyster species, Ostrea chilensis, endemic to New Zealand. The test primers target sequences in the in situ hybridisation probes identified to bind specifically to APX 18S rRNA and amplify a 723 bp DNA product. The test did not amplify 18S rRNA gene sequences of other apicomplexan species, including Toxoplasma gondii, Neospora caninum, Selenidium spp., Cephaloidophorida spp., Lecudina spp. and Thiriotia sp. Of 73 flat oysters identified by histology to be infected with APX at different severities, 69 (95%) tested PCR-positive. Failure to amplify an internal control indicated the presence of PCR inhibitors in the 4 PCR-negative samples. The high analytical sensitivity, specificity and speed of the PCR test should make it a useful tool for detecting APX.

Archigregarines are a key group for understanding the early evolution of Apicomplexa. Here we report morphological, ultrastructural, and molecular phylogenetic evidence from two archigregarine species: Selenidium pygospionis sp. n. and S. pherusae sp. n. They exhibited typical features of archigregarines. Additionally, an axial row of vacuoles of a presumably nutrient distribution system was revealed in S. pygospionis. Intracellular stages of S. pygospionis found in the host intestinal epithelium may point to the initial intracellular localization in the course of parasite development. Available archigregarine SSU (18S) rDNA sequences formed four major lineages fitting the taxonomical affiliations of their hosts, but not the morphological or biological features used for the taxonomical revision by Levine (1971). Consequently, the genus Selenidioides Levine, 1971 should be abolished. The branching order of these lineages was unresolved; topology tests rejected neither para- nor monophyly of archigregarines. We provided phylogenies based on LSU (28S) rDNA and near-complete ribosomal operon (concatenated SSU, 5.8S, LSU rDNAs) sequences including S. pygospionis sequences. Although being preliminary, they nevertheless revealed the monophyly of gregarines previously challenged by many molecular phylogenetic studies. Despite their molecular-phylogenetic heterogeneity, archigregarines exhibit an extremely conservative plesiomorphic structure; their ultrastructural key features appear to be symplesiomorphies rather than synapomorphies.

This study set out to bolster morphological and molecular datasets of marine gregarine apicomplexans. Gregarines were sampled from the Sea of Japan and Northwest Pacific from cirratuliform polychaetes (Acrocirridae, Cirratulidae, and Flabelligeridae), as well as sipunculids. Trophozoites (feeding stages) were gathered for identification using light microscopy, scanning electron microscopy, and transmission electron microscopy. Cells were also collected for molecular phylogenetic analysis using 18S rDNA and 28S rDNA. As a result, three new species of Selenidium, S. planusae n. sp., S. validusae n. sp., and S. pyroidea n. sp. were described, and additional morphological and genetic data were gathered for an existing species, S. orientale; and Trollidium was established as a new genus. Trollidium akkeshiense n. gen. n. sp. possessed a unique, unsymmetrical organization of microtubules running the longitudinal length of one side of the trophozoite, corresponding to a zig-zag pattern of epicytic (surface) folds, and a flicking pattern of movement. Phylogenetic analyses of 18S rDNA and 28S rDNA showed that these portions of the ribosomal operon are able to resolve some relationships among Selenidium, while other lineages including Trollidium akkeshiense n. gen. n. sp. appeared to be highly influenced by long branch attraction. High evolutionary rates along the ribosomal operon of gregarines may hinder this marker from resolving deeper nodes among early apicomplexans.

Recent studies on motility of Apicomplexa concur with the so-called glideosome concept applied for apicomplexan zoites, describing a unique mechanism of substrate-dependent gliding motility facilitated by a conserved form of actomyosin motor and subpellicular microtubules. In contrast, the gregarines and blastogregarines exhibit different modes and mechanisms of motility, correlating with diverse modifications of their cortex. This study focuses on the motility and cytoskeleton of the blastogregarine Siedleckia nematoides Caullery et Mesnil, 1898 parasitising the polychaete Scoloplos cf. armiger (Muller, 1776). The blastogregarine moves independently on a solid substrate without any signs of gliding motility; the motility in a liquid environment (in both the attached and detached forms) rather resembles a sequence of pendular, twisting, undulation, and sometimes spasmodic movements. Despite the presence of key glideosome components such as pellicle consisting of the plasma membrane and the inner membrane complex, actin, myosin, subpellicular microtubules, micronemes and glycocalyx layer, the motility mechanism of S. nematoides differs from the glideosome machinery. Nevertheless, experimental assays using cytoskeletal probes proved that the polymerised forms of actin and tubulin play an essential role in the S. nematoides movement. Similar to Selenidium archigregarines, the subpellicular microtubules organised in several layers seem to be the leading motor structures in blastogregarine motility. The majority of the detected actin was stabilised in a polymerised form and appeared to be located beneath the inner membrane complex. The experimental data suggest the subpellicular microtubules to be associated with filamentous structures (= cross-linking protein complexes), presumably of actin nature.

International audience; Archigregarines, an early branching lineage within Apicomplexa, are a poorly-known group of invertebrate parasites. By their phylogenetic position, archigregarines are an important lineage to understand the functional transition that occurred between free-living flagellated predators to obligatory parasites in Apicomplexa. In this study, we provide new ultrastructural data and phylogenies based on SSU rDNA sequences using the type species of archigregarines, the Selenidiidae Selenidium pendula Giard, 1884. We describe for the first time the syzygy and early gamogony at the ultrastructural level, revealing a characteristic nuclear multiplication with centrocones, cryptomitosis, filamentous network of chromatin, a cyst wall secretion and a 9 + 0 flagellar axoneme of the male gamete. S. pendula belongs to a monophyletic lineage that includes several other related species, all infecting Sedentaria Polychaeta (Spionidae, Sabellaridae, Sabellidae and Cirratulidae). All of these Selenidium species exhibit similar biological characters: a cell cortex with the plasma membrane - inner membrane complex - subpellicular microtubule sets, an apical complex with the conoid, numerous rhoptries and micronemes, a myzocytosis with large food vacuoles, a nuclear multiplication during syzygy and young gamonts. Two other distantly related Selenidium-like lineages infect Terebellidae and Sipunculida, underlying the ability of archigregarines to parasite a wide range of marine hosts.

Although archigregarines are poorly understood intestinal parasites of marine invertebrates, they are critical for understanding the earliest stages in the evolution of the Apicomplexa. Previous studies suggest that archigregarines are a paraphyletic stem group from which other lineages of gregarines, and possibly all other groups of apicomplexans, evolved. However, substantiating this inference is difficult because molecular phylogenetic data from archigregarines, in particular, and other gregarines, in general, are severely limited. In an attempt to help fill gaps in our knowledge of archigregarine diversity and phylogeny, we set out to discover and characterize novel lineages of archigregarines with high-resolution light and scanning electron microscopy and analyses of small subunit (SSU) rDNA sequences derived from single-cell (SC) PCR techniques. Here, we describe two novel species of Selenidium, namely Selenidium idanthyrsae n. sp. and S. boccardiellae n. sp., and demonstrate the surface morphology and molecular phylogenetic position of the previously reported species S. cf. mesnili. We also describe a novel genus of archigregarine, Veloxidium leptosynaptae n. gen., n. sp., which branches with an environmental sequence and, together, forms the nearest sister lineage to a diverse clade of marine eugregarines (i.e. lecudinids and urosporids). This molecular phylogenetic result is consistent with the hypothesis that archigregarines are deeply paraphyletic within apicomplexans, and suggests that convergent evolution played an important role in shaping the diversity of eugregarine trophozoites.

Gregarine apicomplexans inhabit the intestines, coeloms and reproductive vesicles of invertebrates. An emphasis on specific ancestral characteristics in marine gregarines has given the group a reputation of being 'primitive.' Although some lineages have retained characteristics inferred to be ancestral for the group, and perhaps apicomplexans as a whole, most gregarines represent highly derived parasites with novel ultrastructural and behavioral adaptations. Many marine gregarines have become giants among single-celled organisms and have evolved ornate surface structures. A comparison of gregarine morphology, placed in a modern phylogenetic context, helps clarify the earliest stages of apicomplexan evolution, the origin of Cryptosporidium, and specific cases of convergent evolution within the group and beyond.

Cysts and free spores of a metchnikovellid microsporidium were found in several specimens of an archigregarine Selenidium sp. isolated from polychaetes Pygospio elegans. Samples were collected at the littoral area of the Kandalaksha Bay of the White Sea in the year 2016. We examined this material with high-quality light optics in stained and live preparations. The structure of cysts and the host range suggest that this species belongs to the genus Metchnikovella Caullery et Mesnil, 1897. The length of the cysts varied from 9.5 to 34 .m (av. 23.8 .m); the width of the cysts was 4.8–9.2 µm (av. 8.2 µm). The number of cyst-bound spores varied from 7 to 18. Cyst-bound spores were oval or ovoid and arranged in two or three rows. The length of spores was 2.2–3.0 µm (av. 2.6 µm); the width was 1.4–2.9 µm (av. 1.7 µm). Free spores were similar to cyst-bound ones in shape and size. We summarized available data on the species of the genus Metchnikovella. The analysis of these data proved that the shape and size of the examined cysts and the host range of this parasite differ from those of any known species. We named the observed organism Metchnikovella dogieli n. sp.

Archigregarines are dynamic single-celled parasites that inhabit the intestinal systems of marine invertebrates, especially suspension feeding and deposit feeding polychaetes. Certain archigregarines in the genus Selenidium have retained several plesiomorphic characters, and improved knowledge of these species is expected to shed considerable light onto the earliest stages in apicomplexan evolution. Although archigregarines are related to some of the most notorious parasites known (e.g., Cryptosporidium and Plasmodium), current knowledge of the group is meagre. In an attempt to improve our understanding of archigregarine diversity and evolution, I have characterised the general ultrastructure and molecular phylogenetic position of Selenidium serpulae (Lankester) Caullery and Mesnil. The parasites were isolated from the intestines of the calcareous tubeworm Serpula vermicularis (Polychaeta) collected in the eastern Pacific Ocean. The trophozoites (extracellular feeding stages) were spindle-shaped and capable of slow and continuous bending, and coiling, especially when dislodged from the host epithelium. The trophozoite surface was composed of 19–23 longitudinal folds, prominent transverse folds and a robust, trilayered pellicle subtended by a single row of microtubules, each surrounded by an electron transparent sheath. Putative mitochondria were observed, but they were inconspicuous and apparently highly reduced, a condition that is indicative of anaerobic metabolism. The small subunit (SSU) rDNA sequence from S. serpulae was very closely related to two (short) sequences derived from an environmental PCR survey of an oxygen-depleted hydrothermal vent system in the Gulf of California, namely C1-E017 (AY046619) and C2-E016 (AY046806). This result suggested that archigregarines with a morphology and lifecycle much like that in S. serpulae are thriving in this oxygen poor ecosystem. In addition, phylogenetic analyses of a larger SSU rDNA dataset (excluding the shorter environmental sequences) indicated that the nearest sister lineage to S. serpulae was S. vivax, a bizarre tape-like gregarine found in the intestines of sipunculids. This relationship was bolstered by comparative ultrastructural data and helped to illustrate that the diversification and biogeographical distribution of archigregarine parasites is probably more extensive than usually assumed.

Many aseptate gregarines from marine invertebrate hosts are thought to have retained several plesiomorphic characteristics and are instrumental in understanding the early evolution of intracellular parasitism in apicomplexans and the phylogenetic position of cryptosporidians. We sequenced the small-subunit (SSU) ribosomal RNA genes from 2 archigregarines, Selenidium terebellae and Selenidium vivax, and 2 morphotypes of the marine eugregarine Lecudina polymorpha. We also used scanning electron microscopy to investigate the surface morphology of trophozoites from Lecudina tuzetae, Monocystis agilis, the 2 species of Selenidium, and the 2 morphotypes of L. polymorpha. The SSU ribosomal DNA sequences from S. vivax and L. polymorpha had long branch lengths characteristic of other gregarine sequences. However, the sequence from S. terebellae was not exceptionally divergent and consistently emerged as 1 of the earliest 'true' gregarines in phylogenetic analyses. Statistical support for the sister relationship between Cryptosporidium spp. and gregarines was significantly bolstered in analyses including the sequence from S. terebellae but excluding the longest branches in the alignment. Eugregarines formed a monophyletic group with the neogregarine Ophryocystis, suggesting that trophozoites with elaborate cortex folds and gliding motility evolved only once. The trophozoites from the 2 species of Selenidium shared novel transverse striations but differed from one another in overall cell morphologies and writhing behavior.

Three closely related groups of unicellular organisms (ciliates, dinoflagellates and apicomplexans) have adopted what might be the most dissimilar modes of eukaryotic life on Earth: predation, phototrophy and intracellular parasitism. Morphological and molecular evidence indicate that these groups share a common ancestor to the exclusion of all other eukaryotes and collectively form the Alveolata, one of the largest and most important assemblages of eukaryotic microorganisms recognized today. In spite of this phylogenetic framework, the differences among the groups are profound, which has given rise to considerable speculation about the earliest stages of alveolate evolution. However, we argue that a new understanding of morphostasis in several organisms is now throwing light onto the intervening history spanning the major alveolate groups. Insights into the phylogenetic positions of these morphostatic lineages reveal how documenting the distribution of character states in extant organisms, in the absence of fossils, can provide compelling inferences about intermediate steps in early macroevolutionary transitions.

Background Gregarines represent an important transition step from free-living predatory (colpodellids s.l.) and/or photosynthetic (Chromera and Vitrella) apicomplexan lineages to the most important pathogens, obligate intracellular parasites of humans and domestic animals such as coccidians and haemosporidians (Plasmodium, Toxoplasma, Eimeria, Babesia, etc.). While dozens of genomes of other apicomplexan groups are available, gregarines are barely entering the molecular age. Among the gregarines, archigregarines possess a unique mixture of ancestral (myzocytosis) and derived (lack of apicoplast, presence of subpellicular microtubules) features. Methodology/principal findings In this study we revisited five of the early-described species of the genus Selenidium including the type species Selenidium pendula, with special focus on surface ultrastructure and molecular data. We were also able to describe three new species within this genus. All species were characterized at morphological (light and scanning electron microscopy data) and molecular (SSU rDNA sequence data) levels. Gregarine specimens were isolated from polychaete hosts collected from the English Channel near the Station Biologique de Roscoff, France: Selenidium pendula from Scolelepis squamata, S. hollandei and S. sabellariae from Sabellaria alveolata, S. sabellae from Sabella pavonina, Selenidium fallax from Cirriformia tentaculata, S. spiralis sp. n. and S. antevariabilis sp. n. from Amphitritides gracilis, and S. opheliae sp. n. from Ophelia roscoffensis. Molecular phylogenetic analyses of these data showed archigregarines clustering into five separate clades and support previous doubts about their monophyly. Conclusions/significance Our phylogenies using the extended gregarine sampling show that the archigregarines are indeed not monophyletic with one strongly supported clade of Selenidium sequences around the type species S. pendula. We suggest the revision of the whole archigregarine taxonomy with only the species within this clade remaining in the genus Selenidium, while the other species should be moved into newly erected genera. However, the SSU rDNA phylogenies show very clearly that the tree topology and therefore the inferred relationships within and in between clades are unstable and such revision would be problematic without additional sequence data.

Gregarine apicomplexans are a diverse group of single-celled parasites that have feeding stages (trophozoites) and gamonts that generally inhabit the extracellular spaces of invertebrate hosts living in marine, freshwater, and terrestrial environments. Inferences about the evolutionary morphology of gregarine apicomplexans are being incrementally refined by molecular phylogenetic data, which suggest that several traits associated with the feeding cells of gregarines arose by convergent evolution. The study reported here supports these inferences by showing how molecular data reveals traits that are phylogenetically misleading within the context of comparative morphology alone. We examined the ultrastructure and molecular phylogenetic positions of two gregarine species isolated from the spaghetti worm Thelepus japonicus: Selenidium terebellaeRay 1930 and S. melongena n. sp. The ultrastructural traits of S. terebellae were very similar to other species of Selenidium sensu stricto, such as having vermiform trophozoites with an apical complex, few epicytic folds, and a dense array of microtubules underlying the trilayered pellicle. By contrast, S. melongena n. sp. lacked a comparably discrete assembly of subpellicular microtubules, instead employing a system of fibrils beneath the cell surface that supported a relatively dense array of helically arranged epicytic folds. Molecular phylogenetic analyses of small subunit rDNA sequences derived from single-cell PCR unexpectedly demonstrated that these two gregarines are close sister species. The ultrastructural differences between these two species were consistent with the fact that S. terebellae infects the inner lining of the host intestines, and S. melongena n. sp. primarily inhabits the coelom, infecting the outside wall of the host intestine. Altogether, these data demonstrate a compelling case of niche partitioning and associated morphological divergence in marine gregarine apicomplexans.

Selenidium is a genus of gregarine parasites that infect the intestines of marine invertebrates and have morphological, ecological, and motility traits inferred to reflect the early evolutionary history of apicomplexans. Because the overall diversity and phylogenetic position(s) of these species remain poorly understood, we performed a species discovery survey of Selenidium from tube-forming polychaetes. This survey uncovered five different morphotypes of trophozoites (feeding stages) living within the intestines of three different polychaete hosts. We acquired small subunit (SSU) rDNA sequences from single-cell (trophozoite) isolates, representing all five morphotypes that were also imaged with light and scanning electron microscopy. The combination of molecular, ecological, and morphological data provided evidence for four novel species of Selenidium, two of which were established in this study: Selenidium neosabellariae n. sp. and Selenidium sensimae n. sp. The trophozoites of these species differed from one another in the overall shape of the cell, the specific shape of the posterior end, the number and form of longitudinal striations, the presence/absence of transverse striations, and the position and shape of the nucleus. A fifth morphotype of Selenidium, isolated from the tube worm Dodecaceria concharum, was inferred to have been previously described as Selenidium cf. echinatum, based on general trophozoite morphology and host association. Phylogenetic analyses of the SSU rDNA sequences resulted in a robust clade of Selenidium species collected from tube-forming polychaetes, consisting of the two new species, the two additional morphotypes, S. cf. echinatum, and four previously described species (Selenidium serpulae, Selenidium boccardiellae, Selenidium idanthyrsae, and Selenidium cf. mesnili). Genetic distances between the SSU rDNA sequences in this clade distinguished closely related and potential cryptic species of Selenidium that were otherwise very similar in trophozoite morphology.

The trophozoites of two novel archigregarines, Selenidium pisinnus n. sp. and Filipodium phascolosomae n. sp., were described from the sipunculid Phascolosoma agassizii. The trophozoites of S. pisinnus n. sp. were relatively small (64-100 microm long and 9-25 microm wide), had rounded ends, and had about 21 epicytic folds per side. The trophozoites of F. phascolosomae n. sp. were highly irregular in shape and possessed hair-like surface projections. The trophozoites of this species were 85-142 microm long and 40-72 microm wide and possessed a distinct longitudinal ridge that extended from the mucron to the posterior end of the cell. In addition to the small subunit (SSU) rDNA sequences of these two species, we also characterized the surface morphology and SSU rDNA sequence of Selenidium orientale, isolated from the sipunculid Themiste pyroides. Molecular phylogenetic analyses demonstrated that S. pisinnus n. sp. and S. orientale formed a strongly supported clade within other Selenidium and archigregarine-like environmental sequences. Filipodium phascolosomae n. sp. formed the nearest sister lineage to the dynamic, tape-like gregarine Selenidium vivax. Overall, these data enabled us to reassess the molecular systematics of archigregarines within sipunculid hosts and make the following revisions: (1) Filipodium was transferred from the Lecudinidae (eugregarines) to the Selenidiidae (archigregarines), and (2) Platyproteum n. g. was established for Platyproteum vivax n. comb. (ex. S. vivax) in order to account for the highly divergent morphological features and better resolved phylogenetic position of this lineage.

Archigregarines are considered one of the most plesiomorphic groups of Apicomplexa. Until recently, however, this viewpoint was based mainly on the results of the detailed investigation of a single species, Selenidium hollandei. The present study of the fine structure of trophozoites of another archigregarine species Selenidium orientale Bogolepova, 1953. (Apicomplexa, Archigregarinida, as proposed by Grasse and Schrevel), with special reference to the forebody structure of the attached individuals, allows more confident discussion of the plesiomorphic status of the archigregarines. Specimens of S. orientale were collected from the midgut of the Pacific sipunculid Themiste pyroides Chamberlain, 1920. The ultrastructure of the trophozoites generally corresponds to that of other studied species of Selenidium. Differences in the forebody structure between S. orientale and S. hollandei do not conflict with Schrevel's hypothesis on the feeding function of the apical complex in archigregarine trophozoites, although they suggest that, in S. orientale at least, the cytostome is not a persistent structure, but re-opens for each sucking event, and Selenidium trophozoites feed by intermittent sucking of host cytoplasm. Microtubules in the axial zone of the mucron neck may mediate the transport of food vacuoles.

The Gregarinia, all parasitic in invertebrates have appeared in marine hosts (Polychaeta). The most primitive are the Archigregarinida (Selenidium) which are intestinal parasites. The Eugregarinida (intestinal or coelomic) are found in various marine invertebrates as well as in terrestrial or freshwater arthropods. The Neogregarinida which parasitize only insects have a very reduced number of spores compensated by a schizogony (merogony). The Gregarinia constitute a perfect example of a concomitant evolution between a group of parasites and their hosts.

On the basis of a review of the approximately 4300 species of apicomplexan protozoa, the following new species, new names, new combinations, and emendations are given: NEW GENUS, Erhardovina; NEW SPECIES, Ascogregarina polynesiensis, Eimeria golemanskii, Isospora tamariscini; NEW NAME, Gregarina kazumii; NEW COMBINATIONS, Ascogregarina brachyceri (Purrini, 1980), Erhardovina euzeti (Lipa, 1981), E. scutovertexi (Erhardova, 1955), Haemorhormidium batrachi (Chaudhuri & Choudhury, 1983); EMENDATIONS, Selenidium francianum (Arvy, 1952) Tuzet & Ormieres, 1965, Pyxinioides bolitoides D. P. Henry, 1938, P. japonicus H. Hoshide, 1951, P. kamenote H. Hoshide, 1951, P. kurofuji H. Hoshide, 1951, P. oshoroensis H. Hoshide, 1951, P. pugetensis D. P. Henry, 1938, Gregarina levinei Haldar & Sarkar, 1980, Retractocephalus halticae Haldar, Chakraborty & Kundu, 1982, Cnemidospora schizophylli Tuzet & Guerin, 1947, Grebneckiella indica (Merton, 1911) Watson, 1916, Quadruspinospora atractomorphae Haldar & Chakraborty, 1978, Haemogregarina acipenseri Nawrotzky, 1914, H. lobianci Yakimov & Kohl-Yakimov, 1912, H. yakimovikohlae Wladimiroff, 1910, Hepatozoon luehi (Sambon, 1909) Pessoa, Cavalheiro & de Souza, 1970, Eimeria beyerae Ovezmukhammedov, 1977, E. (?) gigantea (Labbe, 1896) Reichenow, 1921, E. (?) labbei Hardcastle, 1943, E. rufi Prasad, 1960, E. (?) scylii (Drago, 1902) Levine & Becker, 1933, Isospora corvi Ray, Shivnani, Oommen & Bhaskaran, 1952, I. melopsittaci Bhatia, Chauhan, Arora & Agrawal, 1973, I. seicerci Ray, Shivnani, Oommen & Bhaskaran, 1952, I. stomatici Chakravarty & Kar, 1944, I. triffitae Nukerbaeva & Svanbaev, 1973, Wenyonella mackinnonae Misra, 1947, Octosporella sanguinolentae Ovezmukhammedov, 1975, Lankesterella millani Alvarez Calvo, 1975, Sarcocystis woodhousei Dogel', 1916, Haemoproteus lari Yakunin, 1972, Babesia ninakohlyakimovae (Yakimoff & Shokhor, 1916), Theileria ninakohlyakimovae (Yakimov, 1916) Krylov, 1974, Haemohormidium batrachi (Chaudhuri & Choudhury, 1983).

SYNOPSIS. The taxonomic position of Selenidiidae Brasil in the class of gregarines is discussed in relation to the study of its life cycles, its possible schizogony and the fine structure of its trophozoites. The cycle of gregarines which belong to the genus Selenidium Giard is characterized by trophozoites with pendular or coiling movements, nuclear transformations in gamonts during syzygy just before cyst formation, anisogamy, sporocysts with 4 sporozoites. Schizogony of the Selenidiidae is not yet demonstrated. The “kystes a meerozoites” within the gut epithelium of Sabellaria alveolata, could be one of the stages of schizogony of S. hollandei. This sole example in our study and the absence of schizogony in numerous species, especially in S. pendula, the type species, shows that this criterion is uncertain, actually, in the definition of the order Archigregarinida Grasse. Ultrastructural studies of S. hollandei and S. pendula show that the cortical region in trophozoites of the genus Selenidium is different from that of Eugregarinida. In the Selenidiidae the epicyte is composed of longitudinal folds. Under the wall, consisting of 3 membranes there is a well-defined pellicular fibrillar system. In S. hollandei, the trophozoite has a fibrillar formation, corresponding to a conoid in its anterior region. The trophozoites of S. hollandei and S. pendula contain anterior dense bodies or rhoptries which are very well developed. All these characteristics conform to the ultrastructural organization of the dissemination forms (merozoites, schizozoites, sporozoites): The results allow one to give a new definition of the Order Archigregarinida: Order Archigregarinida (Grasse): Gregarines with ultrastructural organization of the trophozoites similar to that of dissemination forms. Presence of a well defined pellicular fibrillar system. Intestinal parasites of polychaete worms.

SUMMARY. The author proposes a new systematic division of the suborder Schizogregarina on the basis of the comparative morphology of the different stages of the life-cycle of the different genera. Two families, Dischizae and Monoschizae, are divided into a total of 6 tribes: The Dischizae into Ophryocystinae with the genera Ophryocystis, Mattesia, Menzbieria, Lipocystis and Sawayella, and Machadoellinae with the genera Machadoella and Farinocystis; Monoschizae into the tribe Caulleryellinae with genera Caulleryella, Tipulocystis and Merogregarina, the tribe Syncystinae with the genera Syncystis and Lipotropha, the tribe Schizocystinae with the genera Schizocystis and Siedleckia and the tribe Selenidiinae with the genera Selenidium and Meroselenidium.

1. A detailed description is given of a coccidian which parasitises the nuclei of the intestinal cells of the polychaete worm Polydora flava Claparède. The final stages in sporogony have not been observed; nevertheless, this is the first record, from an annelid worm, of an eimeriid which shows a striking resemblance to those Coccidia occurring in the higher animals. It is also the first record of an intranuclear parasite from an annelid.2. From the gut of the same worm, a new species of Selenidium is described. It differs from all other species, hitherto recorded, in the possession of an axial tube, which completely encloses the nucleus and extends from one end of the organism to the other: hence it is proposed to name this organism Selenidium axiferens. The anterior end of the trophozoite shows a circlet of siderophilous bodies, which Ray (1930 a) has shown to be characteristic of the genus.3. Both uninucleate and multinucleate intracellular forms, with polar groups of chromatic globules, have been discovered in great abundance on six occasions; no merozoites have been found, but evidence is submitted to show that these intracellular forms represent the schizogonic phase in the life-cycle of the Selenidium.4. Sporogony follows closely that described for other species, but the final stages have not been observed. The gametocysts measure 48 × 50–70 × 63μ.5. Association and cyst formation are described in Polyrhabdina polydorae Caul. & Mesn. (?) for the first time. Innumerable nuclei make their appearance in each gametocyte, before ejection of the cysts into the sea-water takes place. Gametocysts can easily be distinguished from those of Selenidium axiferens, not only by their morphology, but also by their greater size (98 × 90–105 × 100μ).

In 1930 Ray published a valuable paper on the genus Selenidium Giard, in which he described sporogony in S. spionis Koll. and S. foliatum Ray. He did not observe schizogony in either of these two, but he confirmed Brasil's account of intracellular schizogony in S. caulleryi Brasil from Protula tubularia Mont, and recorded it for the first time in S. mesnili Brasil from Myxicola infundibulum Mont. After seeing association of gametocytes in both these species he was unable to continue his observations, so I have taken up the work where he left it, choosing S. mesnili rather than S. caulleryi, since it is more easily obtainable. My search for gamete and sporozoite formation has been successful, but I have not been able to make the sporozoites hatch, or in studying sections of Myxicola have I seen sporozoites entering the epithelial cells of the gut.

SYNOPSIS. In S. fallax and D. cirratuli the pellicle is thrown into longitudinal folds. The pellicle consists of 2 membranes, one 3-layered, the other 5-layered. Sub-pellicular fibrils, each about 23 mμ in diameter run along the pellicular folds. Pellicle and sub-pellicular fibrils together form the mechanism by which Selenidiids move. The undulating membranes of D. cirratuli are enlarged pellicular folds, the tips of which are packed with fibrils. The light ovoid patches seen in thin sections are sections through regions which contain a form of glycogen in the living animals. Typical mitochondria are absent. Objects described as lipochondria have a regular distribution at the bases of the grooves in the pellicle and are probably formed from the inner components of the pellicle.

SYNOPSIS. The gregarine order Archigregarinorida is redefined. The new family Selenidioididae is established in it, containing the genera Selenidioides n. g. with 11 species, Meroselenidium with 1 and Merogregarina with 1. The new family Exoschizonidae is also established in it, with 1 genus, Exoschizon, containing 1 species. The family Selenidiidae is transferred to the order Eugregarinorida in a new superfamily, Lecudinicae, and redefined to exclude the presence of merogony. As redefined, the family Selenidiidae contains the genera Selenidium with 33 species, Selenocystis with 1 and Ditrypanocystis with 2. The following new species are named: Selenidioides fanthami, S. hawesi, Selenidium mackinnonae, S. martinensis, S. rayi, Ditrypanocystis coxi. The following new combinations are introduced: Selenidioides axiferens, S. caulleryi, S. giganteum, S. hollandei, S. intraepitheliale, S. mesnili, S. metchnikovi, S. potamillae, S. sipunculi, Selenidium synaptae. In addition, the septate form from Cirratulus cirratus that was named Polyrhabdina cirratulus by Bogolepova (2) is renamed P. bogolepovae n. sp.

1. The main facts in Brasil's (1907) account of the intracellular schizogony inSelenidium caulleryifromProtula tubulariahave been confirmed. Early stages in the sporogony of this species are described now for the first time.2. Intracellular schizogony is described for the first time inSelenidium mesniliBrasil fromMyxicola infundibulum, and here also the early stages in sporogony are noted for the first time.3. The life histories of Caullery and Mesnil's two unnamed species ofSelenidium(now calledS. spionis(Köll.) andS. foliatumn.sp.) fromScolelepis fuliginosahave been exhaustively studied. No schizogonic phase has been observed in either. The gametocysts, here described for the first time, are found to develop fully only after escape into the sea. The gametocysts ofS. spionisare ovoid, 108μ x 60μ and contain spores with four sporozoites: those ofS. foliatumare spherical, 70μ in diameter, and their spores contain eight sporozoites.4. New or hitherto little known species ofSelenidiumare recorded fromCirratulus cirratusMüll.,Branchiomma vesiculosumMont.,Sabella pavoninaSav.,Pomatoceros triqueterL. andTerebella lepidoriaKühl, and the morphological characters of the trophozoites are described.5. A discussion follows of the value of the diagnostic characters of the genusSelenidium. It is suggested that this genus requires drastic revision and will probably have to be dismembered. Stress is laid, however, on the occurrence in all the gregarines examined, and at all the observed stages of their development, of characteristic chromatic bodies at the anterior end, structures which have hitherto escaped the notice of most observers.

1. The term Schizogregarinae Léger (1900) is the name now given to a sub-order of the Gregarinida, the remaining members of which are known as the Eugregarinae. The Schizogregarines were formerly known as Amoebosporidia Aimé Schneider (1884), a name given in misapprehension of the character of the cytoplasmic processes, fixative in function, present in the genus Ophryocystis. Two species of Ophryocystis (0. buetschlii and 0. francisci) were the only members of this sub-order known before 1900.2. At present the sub-order Schizogregarinae contains five families: Ophryocystidae, Schizocystidae, Selenidiidae, Merogregarinidae, and Aggregatidae.3. All these organisms show well-marked schizogonic stages in their life-history, and—with the possible exception of the Aggregatidae—follow after the Eugregarinae in their methods of sporogony.4. In Ophryocystis and Schizocystis the schizogony is extracellular, that is, these forms are ectoschizous. The life-cycle of the former is shown in Fig. 1. In these parasites the number of the nuclei in the schizont increases simultaneously with its volume.5. In Selenidium and Merogregarina the schizogony is intracellular, in other words these forms are endoschizous. The life-cycle of the former is illustrated in Fig. 3. In these forms the schizont is uninucleate during its growth, only becoming multinucleate at the end of the growing period.6. Ophryocystis forms only one sporocyst, a fact which has been emphasised by Léger and Duboscq (1908), by the placing of the Ophryocystidae in a special section, the Monospora. However, this apparent peculiarity is easily explained by a process of reduction and degeneration having taken place, affecting with one exception all the gametes formed from each gametocyte. There is good morphological evidence in support of this explanation (see p. 382, and Fig. 4, D).7. Figures of the interesting form Schizocystis gregarinoides (Léger, 1900) are not yet published, but a paper dealing with this organism is promised by Prof. Léger at an early date.8. Aggregata differs from other Schizogregarines in that its schizogony takes place in one host (crab), while its sporogony occurs in another (Cephalopod mollusc). In this respect Aggregata resembles the Haemosporidia. The schizogonic phases in Crabs were formerly regarded as belonging to a gymnosporous Gregarine, Aggregata Frenzel, while the sporogonic phases were considered to belong to a Coccidian, Eucoccidium (Benedenia) in cuttlefishes and Octopus. Regarding this, Léger and Duboscq (1908, p. 102) write “…Aggregata, avec un changement de cycle coïncidant avec un changement d'hôte, c'est à dire qui soient à la fois digénétiques et hétéroïques.”9. It is evident therefore that the Aggregatidae stand apart. On this account, I suggest a division of the Schizogregarinae into two sections, termed respectively, the Homoïica (to include the first four families discussed in this paper, wherein schizogony and sporogony take place in the same host) and the Heteroïca (for the Aggregatidae).10. Among the Homoïca we have extracellular schizogony (ectoschizous forms) in the Ophryocystidae and Schizocystidae, and intracellular schizogony (endoschizous forms) in the Selenidiidae and Merogregarinidae. This difference is not merely superficial, it requires to be emphasised, and for this reason I would divide the section Homoïca into two sub-sections, termed respectively Ectoschiza and Endoschiza.11. The classification of the Schizogregarines, which I would propose, is as follows:12. Much further research is needed on the life-cycles of the Endoschiza, especially among the Selenidiidae, which occur so frequently in the Annelida. Sporogonic stages are at present unknown in Eleutheroschizon and Siedleckia.13. In connection with the Aggregatidae, and to a less extent with the Selenidiidae, stress is laid upon the necessity of carefully distinguishing between “coelomic” and “gut” parasites. (See pp. 397 and 387.)14. The Schizogregarinae form a most interesting link between the Eugregarinae and the Coccidiidea.

1. Two new schizogregarines are described from the gut of the polychaete worm Potamilla reniformis (O.F.M.). One of these is meanwhile retained in the genus Selenidium Giard as S. potamillae n.sp. For the other a new genus, Meroselenidium, is proposed, of which M. keilini is the type species. The life cycle has been followed in both gregarines.2. The schizogony of Meroselenidium is characterised by the formation of cytomeres preliminary to the liberation of merozoites. In Selenidium potamillae the schizogony is more like that in S. mesnili and S. caulleryi, though there is a considerable residual body.3. The gametocytes become attached by their posterior ends. In S. potamillae there is often a multiple association. The gamete nuclei are formed within the nuclear area of the gametocytes. Further development takes place in sea water. Isogametes have been observed in S. potamillae.4.The gametocyst of Meroselenidium keilini is elliptical in outline and measures about 70 × 55μ. It contains some twenty relatively large spores, 27 × 15μ, the walls of which are transversely ridged; the spores are polyzoic. The gametocyst of S. potamillae is of much the same shape, but only about half the size. The sporocysts are very numerous, spherical, smooth-walled, and measure about 8·5μ in diameter. In neither species does the gametocyst contain any residual mass.We take this opportunity of thanking the High Commissioner of India and the Delegacy of King's College for grants-in-aid received by one of us (H. N. R.) during the course of these investigations.

The trophozoites of Selenidium fallax propagate bending waves at rates of up to 35 μm s−1, of a similar character to those manifested by eukaryotic cilia and flagella. A beat frequency of 0·12–0·15 Hz appears average, though rates outside this range have been recorded. Translatory locomotion at up to 6 μm s−1 has been observed. The protozoan demonstrates the presence of an active bending mechanism, probably along its entire length, and a means of coordinating adjacent bends. The Reynolds number for the motion is 10−5-10−4, suggesting that the hydrodynamic aspects of the trophozoite movement are amenable to analysis by similar means to those already employed for cilia and flagella. It is possible that the protozoans exhibit a sliding microtubule mechanism, which could be very usefully compared with that occurring in the ciliary axoneme.

SYNOPSIS. The fine structure of the eugregarine Pyxinoides balani was studied and compared with that of other gregarines previously examined with the electron microscope. P. balani contains cell organelles comparable to those in the archigregarine Selenidium hollandei. These include mitochondria, Golgi complexes, granular and agranular endoplasmic reticulum, membrane-limited granules, vacuoles and myonemes. A comparison of the distribution and fine structure of these organelles with those of the above archigregarine was made. The possible function of the Golgi complex in assisting in producing the transverse septum is suggested. The surface of P. balani is composed of a surface-membrane complex different from that of S. hollandei, and similar to those of the eugregarines previously studied. The complex is composed of an outer plasmalemma, subjacent pentalaminar layer, and underlying, homogeneous, electron-dense layer. The pentalaminar layer is 140–170 A thick and has a structure reminiscent of fused cell membranes seen in tight junctions, myelin figures and nerve myelin. The pentalaminar layer is derived from subsurface cisternae found in sporozoites. The underlying, homogeneous, electron-dense layer is 450–500 A thick. Pores and caveolae are found as specializations of the pentalaminar and homogeneous electron-dense layers. Endoplasmic reticulum is closely associated with the caveolae. The location of the pentalaminar layer at the surface and the presence of pores and caveolae in its structure suggests some transport and/or segregation function.

SYNOPSIS Gregarine Selenidium fauchaldi sp. n. is described from the intestinal lumen of the polychaete Phragmatopoma californica (Fewkes, 1889) in the intertidal zone of the Pacific Ocean on Santa Catalina Island, California. In addition, the new combination Selenidium telepsavi (Stuart, 1871) comb. nov. is introduced for the gregarine originally described as Monocystis telepsavi from the polychaete Telepsavus castarum in the Black Sea.

Selenidium vivax n. sp. (Protozoa, Apicomplexa, Eugregarinorida, Selenidiidae) is described from the intestine of the sipunculid Phascolosoma agassizii from the central Califomian coast. Gamonts were up to 550 Am long, flat, variable in shape, with an asymmetric rostrum and without permanent surface striations.

An orthonectid parasitizing the sabellariid polychaete Sabellaria cementarium Moore has been studied with the aid of a method of impregnation with activated silver albumose (Protargol). This technique differentiates the boundaries of many of the cells, and also brings out the kinetosomes of cilia, thereby permitting a precise analysis of the pattern of ciliation. In adult individuals, the cells of the outer jacket are arranged in rings around the axial mass. Certain of the jacket cells are characterized by presence of conspicuous granules and at least one transverse row of cilia, whereas others have neither obvious granules nor cilia. In both sexes, the first three rings of jacket cells bear three transverse rows of cilia; all other ciliated cells have either one row of cilia near the posterior margins or a row near both the anterior and posterior margins. In typical males, there are 26 or 27 rings, of which rings 6, 10, 12, 15, 17, 20, and 22 are unciliated. In typical females, there are 36 or 37 rings, of which rings 6, 8, 10, 12, 14, 16, and 18 are unciliated. In the male, the mass of gametes or prospective gametes occupies the posterior half of the axial mass. In the female, there is a column of 9 to 12 oocytes occupying the posterior four-fifths of the axial mass. The name Ciliocincta sabellariae gen. n., sp. n. is proposed for this orthonectid. The type locality is near Dot Rock (long 122047.8/ W; lat 48?29.5' N), San Juan Archipelago, Washington. Of 88 worms of the host species examined from this locality, only two (both males) were infected. The few known species of orthonectid mesozoans are parasites of acoel, rhabdocoel, and polyclad turbellarians, nemerteans, polychaete annelids, gastropod and pelecypod molluscs, and ophiuroid echinoderms. Except for one report of an orthonectid parasitizing a nemertean collected near Woods Hole, Massachusetts (Meinkoth, 1956), all published accounts deal with species occurring in the Mediterranean Sea or in the Atlantic waters of Europe. That these interesting organisms will eventually be found in a variety of hosts in North America and elsewhere may confidently be predicted, but attempts to find them on the Pacific coast in invertebrates of the same taxonomic groups as those which they are known to parasitize have until recently been unsuccessful. My discovery of orthonectids, at the Friday Harbor Laboratories of the University of Washington, came about in a nearly fortuitous manner. On 14 July 1962 I collected a large number of brittle stars (Ophiura sarsi) with a view to examining them for orthonectids. After disReceived for publication 31 August 1964. 37 secting over 80 specimens with negative results, I liberated the rest of the catch. Returning to the laboratory, I decided to examine a few specimens of the polychaete Sabellaria cementarium Moore (Sabellariidae) which had been collected on the same dredging trip. From previous experience, I knew that this species was regularly infected by a so-called schizogregarine of the genus Selenidium. As the first worm was opened, hundreds of male and female orthonectids emerged from the tissue of the body wall. Over a period of several days, I examined a total of 88 worms (47 males, 41 females) from the same locality. The sixtysixth specimen to be opened also was infected. Both of the parasitized worms were mature males carrying sperm, and before examination appeared to be completely normal. The orthonectid from S. cementarium, for which I propose the name Ciliocincta sabellariae gen. n., sp. n., resembles certain species of Rhopalura. However, its precise pattern of ciliation is decidedly different from that described by any of the European investigators. It is possible that if the morphology of other This content downloaded from 207.46.13.114 on Thu, 26 May 2016 06:30:46 UTC All use subject to http://about.jstor.org/terms 38 THE JOURNAL OF PARASITOLOGY, VOL. 51, NO. 1, FEBRUARY 1965 orthonectids were investigated carefully, some of them would be found to have an organization similar to that of C. sabellariae. The type locality for C. sabellariae is near Dot Rock (long 122o47.8' W; lat 48?29.5' N), San Juan Archipelago, Washington. The material was collected at a depth of 7 fathoms. Syntype slides have been deposited in the United States National Museum (USNM No. 23734).